9502
Biochemistry 1992, 31, 9502-9512
Membrane Stimulation of cGMP Phosphodiesterase Activation by Transducin: Comparison of Phospholipid Bilayers to Rod Outer Segment Membranes? Justine A. Malinski and Theodore G. Wensel’ Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030 Received January 28, 1992; Revised Manuscript Received May 28, 1992
ABSTRACT: To clarify the role of phospholipids in G protein-effector interactions of vertebrate phototransduction, transducin activation of cGMP phosphodiesterase (PDE) has been reconstituted on the surface of well-defined phosphatidylcholine (PC) vesicles, using purified proteins from bovine rod outer segments (ROS). PC vesicles enhanced PDE stimulation by the GTP-yS-bound transducin a subunit (T,-GTPyS) as much as 17-fold over activation in the absence of membranes. In the presence of 3.5 p M accessible PC in the form of large (100 nm) unilamellar vesicles, 500 nM T,-GTPyS stimulated PDE activity to more than 70% of the maximum activity induced by trypsin. Activation required PC, PDE, and T,-GTPyS, but did not require prior incubation of any of the components, and occurred within 4 s of mixing. The PC vesicles were somewhat more efficient than urea-washed R O S membranes in enhancing PDE activation. Half-maximal activation occurred at accessible phospholipid concentrations of 3.8 pM for PC vesicles, and 13 pM for R O S membranes. Titrations of PDE with T,-GTPyS in the presence of membranes indicated a high-affinity (Kacc< 250 pM) activation of PDE by a small fraction ( 0 5 5 % ) of active T,-GTPyS, as did titrations of R O S with GTPyS. When activation by PC vesicles was compared to PDE binding to membranes, the results were consistent with activation enhancement resulting from formation of a T,GTPy S-dependent PDE-membrane complex with half-maximal binding at phospholipid concentrations in the micromolar range. The value of the apparent dissociation constant, KPL,associated with the activation enhancement was estimated to be in the range of 2.5 nM (assuming an upper limit value of 1600 phospholipids/ site) to 80 nM (for a lower limit value of 50 phospholipids/site). Another component of membrane binding was more than 100-fold weaker and was not correlated with activation by T,-GTPyS. Low ionic strength disrupted the ability of R O S membranes, but not PC vesicles, to bind and activate PDE. Removal of PDE’s membrane-binding domain by limited trypsin digestion eliminated both the binding of PDE to vesicles and the ability of PDE to be activated by T,-GTPyS and membranes. These results suggest that ROS membrane stimulation of PDE activation by T,-GTPyS is due almost exclusively to the phospholipids in the disk membrane.
Transducin and cGMP phosphodiesterase (PDE’) are peripheral membrane proteins found on the disk membranes of vertebrate rod cells [for reviews, see Stryer (1986, 1991), Liebman et al. (1987), and Hurley (1987)l. In its active, GTP-bound form, the a subunit of transducin (T,-GTP) activates PDE by reversing the effect of the PDE inhibitory subunit, PDE, (Hurley & Stryer, 1982; Wensel & Stryer, 1986). While T,-GTP and PDE, are known to act in reciprocal fashion to regulate the catalytic subunits (PDE,B), the composition and stoichiometry of the complex(es) formed by T,-GTP and PDE subunitsand the relationship of the proteins This work was supported by National Institutes of Health Grants EY07981 and EY06255. Abbreviations: PDE, photoreceptor cGMP phosphodiesterase, PDE,8,,; T, transducin, the photoreceptor G protein; T, and Tp,, a and subunits of transducin; T,-GTPyS, activated transducin, GTPySbound a subunit;PC, phosphatidylcholine;LUV, large unilamellarvesicles, approximately 100 nm in diameter; MLV, multilamellar vesicles; Kact, the apparent dissociation constant for the interaction of T,-GTPyS with , apparent dissociation PDE in the presence of a membrane; n K p ~ the constant for the T,-GTP-/S-PDE activated complex and the phospholipid bilayer, defined as the product of the number of phospholipid molecules constituting an individual binding site ( n ) and the apparent dissociation constant for that site ( K P L ) R, ; total photochemical products of bleached rhodopsin; ROS,rod outer segments; UW ROS,urea-washed rod outer segment membranes;GTPyS, guanosine 5’-[y-thio]triphosphate;MOPS, 4morpholinepropanasulfonicacid; Tris, tris(hydroxymethy1)aminomethane; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; DTT, dithiotreitol; PMSF, phenylmethanesulfonyl fluoride.
0006-2960/92/043 1-9502$03.00/0
to the membrane when PDE is activated have yet to be determined with certainty. The affinity of T, for membranes is greatly reduced in the GTP form, and light scatteringsignalsfrom rod outer segments have been interpreted as indicating the rapid release of T,GTP from membranes triggered by light activation of rhodopsin (Kahn et al., 1981; Lewis et al., 1984;Vuong et al., 1984). Despite the increased solubility of T, in the activated form, it only activates PDE efficiently in the presence of membranes (Fung & Nash, 1983;Ho & Fung, 1984;Tyminski & O’Brien, 1984;Wensel & Stryer, 1986; Miller et al., 1987; Bennett & Clerc, 1989; Phillips et al., 1989), implying a requirement for a membrane-boundcomplex to form activated PDE. While the solubilization of T, upon binding GTP is unique among characterized G proteins, the localization of G protein-ffector interactions at the cytoplasmic surface of membranes is a common characteristicof all known G proteineffector interactions. In order to determine the molecular mechanism for this membrane-mediated activation, it is essential to be able to reconstitute the activation process from well-defined purified components whose concentrationscan be controlledand whose participation in complex formation can be accurately determined. Activation of purified PDE by purified transducin has been reconstituted on membranes, but previous studies have used membranes containing significant amounts of other proteins [primarily rhodopsin; e.g., Fung and Nash (1983), Tyminski and O’Brien (1984), Wensel and Stryer (1986), 0 1992 American Chemical Society
T,-GTPyS and Phospholipid Activation of cGMP PDE Miller et al. (1987), and Bennett and Clerc (1989)] or of positively charged detergents (Ho & Fung, 1984) or have resulted in only weak membrane enhancement of PDE activation (Phillips et al., 1989). We have sought to determine whether the efficient activation of PDE by activated T, observed in the presence of disk membranes can be obtained using protein-free bilayers of purified phospholipids and to characterize the requirements for efficient reconstitution. We report here a comparison of the ability of well-defined phosphatidylcholine vesicles and bovine rod outer segment membranes to serve as an activation surface, Le., to bind activated T, and PDE and to promote enhanced T,-GTPyS activation of PDE. MATERIALS AND METHODS
Reagents. GTPyS, MOPS, and DTT were obtained from Boehringer-Mannheim. EDTA was from Fisher Scientific. Reagents for electrophoresiswere from Bio-Rad. Frozen darkdissected bovine retinas were obtained from J. A. Lawson Packing (Lincoln, NE). Other reagents were obtained from the Sigma Chemical Co. Buffers. The moderate-saltbuffer contained 10mM MOPS (pH 7.4), 30 mM NaC1,60 mM KCl, 2 mM MgC12, 1 mM DTT, and solid PMSF. The low-salt buffer contained 5 mM Tris (pH 7.4), 0.5 mM MgC12,l mM DTT, and solid PMSF. The pH assay buffer contained 20 mM MOPS (pH 8.0), 150 mM KCI, 2 mM MgC12, and 0.1 mM EDTA. Subunit separation buffer contained 10 mM MOPS (pH 7.4), 5 mM MgC12, 0.1 mM EDTA, and solid PMSF. All buffers were sterile filtered through 0.2-pm filters (Sartorius) immediately prior to use. Rod Outer Segments and Protein Preparation. ROS were prepared under dim red light from frozen dark dissectedbovine retinas as described (Fung & Stryer, 1980). ROS membranes stripped of peripheral proteins (UW ROS) were prepared by washing with 4 M urea (Yamanaka et al., 1985). These membranes were tested by papain treatment (Fung & Hubbell, 1978) toverify that >90% of R was accessibleto the protease, and it is therefore assumed that approximately 50% of the total phospholipid was accessible to added proteins. Preparation of transducin by GTP elution of hypotonically washed ROS and chromatography on hexyl-agarose was carried out as described (Fung et al., 1981). Activated transducin, T,-GTPyS, was prepared either from holotransducin or by direct elution with GTPyS from low-salt-washed ROS. Activation of holotransducin (25 pM) was carried out in subunit separation buffer with 5 pM bleached rhodopsin in UW ROS and 10 pM GTPyS (3-mL total volume). The mixture was incubated at 4 OC for 2 h followed by removal of the membranes by centrifugation at 44 000 rpm in a Beckman TLA 100.3 rotor for 15 min at 4 OC. The volume of the supernatant containing the activated transducin was increased to 20 mL with subunit separation buffer and applied to a Reactive Blue 2-Sepharose CL-6B (Blue Sepharose) column (3 cm X 7 cm) equilibrated in the same buffer at 4 OC. The column was washed and the by subunit eluted in subunit separation buffer supplemented with 100 mM KC1 (150 mL) followed by elution of the CY subunit in the same buffer with 750 mM KCl(l50 mL), Fractions were collected (5 mL) and analyzed by SDS-PAGE. The pooled proteins were concentrated by ultrafiltration under 50 psi N2 with YMlO filters (Amicon) at 4 "C. All purified proteins were stored in 50% glycerol (v/v) at -20 "C. Alternatively, T,GTPyS was prepared by GTPyS (10 pM) extraction of washed ROS, followed by chromatography on Blue Sepharose as
Biochemistry, Vol. 31, No. 39, I992 9503
described above. T,-GTP$ prepared on Blue Sepharose contained less than 1 PDE/3000 T,-GTPyS. The T,-GTPyS used in the direct-binding studies was prepared by mixing holotransducin with equimolar R (in urea-washed ROS membranes), in pH assay buffer, removing glycerol and unbound transducin by centrifugation, and eluting T,-GTP-/S and some TB, from the membranes with pH assay buffer containing GTPyS. The protein was kept on ice and used within 24 h. PDE was prepared essentially as described by Wensel and Stryer (1986,1990). Bleached ROS membranes (300retinas) were extracted with moderate- and then low-salt buffers. The low-salt extract was adjusted to 30 mM sodium phosphate (pH 7.2), 50 mM NaCl, and 25% (v/v) glycerol and applied to a 3 cm X 7 cm column of hydroxylapatite (Bio-Rad) equilibrated in the same buffer at 4 OC. The column was washed and the PDE was eluted with three stepwise increases in phosphate concentration: 80, 150, and 300 mM (2:l Na+ to K+, pH 7.2) 25% in glycerol (v/v), 100-150 mL each. P D E s membrane-binding domain was removed by incubating PDE wtih 0.01 mg/mL trypsin for 5 min (Figure 3A) or with 0.005 mg/mL trypsin for different times up to 20 min at room temperature. The reaction was stopped by adding soybean trypsin inhibitor to an inhibitor to trypsin ratio of 10 (w/w). Control samples were prepared by adding PDE to trypsin preincubated with inhibitor. Phospholipid Vesicles. All vesicles were prepared with highly purified phospholipids. PC (egg yolk) was purchased from the Sigma Chemical Co. Large unilamellar vesicles (LUV) were prepared by modification of an extrusion method (Hope et al., 1985; Mayer et al., 1986). Organic solvent was removed from the phospholipid stocksolution, 20-40 mg, under a gentle stream of dry N2, and residual organic solvent was removed under reduced pressure for at least 2 h. The dried phospholipid film was resuspended in 1-2 mL of moderatesalt buffer with 0.1 mM EDTA, 1 mM DTT, and 0.05%NaN3 (w/v) by vigorous agitation. The suspension was subjected to six freeze-thaw cycles in alternating dry iceacetone and warm water baths and then forced, at least 10 times, through two stacked 0.1-pm polycarbonate filters (Costar/Nuclepore, Pleasanton, CA) encased in a 25-mm Millipore high-pressure stainless steel filter holder (XX45-025-00) coupled to a Waters HPLC pump with a 2-mL injection loop. The size of vesicles prepared in this manner was determined by quasi-elastic light scattering using a Langley/Ford LSA2 light scattering spectrophotometer coupled to a Model 1096 correlator (Bloomfield & Lim, 1978). Diffusion constants were converted to hydrated radii by the Stokes-Einstein equation. The diameter of the PC LUV measured 103.8 f 2.1 and 99.4 f 2.2 nm, for two autocorrelation time constants which differed by a factor of 2, indicating a homogeneous size distribution. LUV (100 nm) prepared using other extrusion devices after repeated freeze-thaw cycles have been shown to be predominantly unilamellar. That the vesicles made with our modified procedure were unilamellar was confirmed by 31PNMR. The experimentswere performed on a Bruker AM-400 (wide band) NMR spectrometerequipped with an Aspect-3000data system The spectra, 8000 data points/ operating at 161 MHz (31P). scan with a spectral width of 16 kHz, were acquired in the unlocked mode with broad-band proton decoupling at 40 OC and externally referenced to 80% polyphosphoric acid. The 31Pspectrum of phosphate in phospholipid bilayers consists of a single resonance which can be split into two lines by addition of Pr3+ ions. One peak remains at the original
9504 Biochemistry, Vol. 31, No. 39, 1992
Malinski and Wensel
resonanceand the other is paramagnetically shifted downfield, representingthe phosphate inaccessible and accessible to Pr3+, respectively (Brainard et al., 1980). Theratiooftheintegrated areas for these two lines was 0.99, indicating that about half of the phosphorus was accessible to the Pr3+ions, Le., located in the external leaflet, and half was not. The multilamellar vesicles (MLV) used in binding experiments were prepared in an identical manner except that the aqueous suspension was forced through two 0.4-pm filters. Because of their large size, nearly all of such vesicles are multilamellar (Mayer et al., 1986). To ensure quantitative separation of PDE bound to the vesicles from PDE free in solution, the most rapidly sedimenting vesicles were selected by centrifugation far 10 min in a microcentrifuge ( 13000g). The supernatant containing the smaller vesicles was removed, and the pellet was resuspended in a small amount of buffer (0.5-1 mL). The resuspendedpellet fraction, designated 400nm MLV, was the source of the phospholipid vesicles used in direct-binding experiments. The fraction of external phospholipid was determined with 31PNMR as described above. For PC MLV the ratio of accessible to total phosphorus was 0.28. All phospholipid concentrations for PC vesicles and ROS membranes are expressed in terms of accessible phospholipid, as are apparent dissociation constants calculated from titration data. PDE Assays. PDE activity was measured by continuously monitoring the amplified signal from a pH electrode (MI4 10, Microelectrodes, Inc., Londonderry, NH) indicating the release of protons from cGMP (Liebman & Evanczuk, 1982), using 2 mM cGMP unless noted, at room temperature, in pH assay buffer with 0.1%ovalbumin (w/v) in a findl volume of 200 pL. The measured hydrolysis rate was found to be linear with the concentration of active PDE throughout the range of total PDE activity used in these experiments. Reconstitution of Tu-GTPyS- and membrane-stimulated PDE activity was accomplished by adding the purified proteins and phospholipid vesicles or UW ROS membranes individually from concentrated stocks to the stirred solution. Total PDE activity in the sample was defined as the activity measured followingaddition of excess trypsin (typically, 0.25 mg/mL). PDE-membrane binding was directly determined in sedimentationexperiments. PDE was mixed with the indicated amounts of membranes in the presence or absence of T,-GTPyS (2 pM) in pH assay buffer to a final volume of 175 pL and a final glycerol concentration of0.896 or less. PDE that bound the membranes was separated from free PDE by sedimentation in an airfuge, 28 psi (- 130000g), for 20 min at room temperature. The amount of PDE present in the supernatant and pellet fractions was measured by activity following trypsin activation. GTPyS activation of PDE in native ROS membranes was determined by the pH method at room temperature. Sufficient GTPyS to give the indicated final concentrations was added to freshly bleached ROS membranes, 2 pM R, in pH assay buffer. In order to estimate the uncertainty in activity measurements, we compared 23 independent measurements of trypsinstimulated activity of PDE on 3 different days over a 7-day period. The activity for a nominal concentration of 10 nM PDE was found to be 39.5 pM cGMP hydrolyzed/s, with a standard deviation of f 3.4 pM/s. In general, deviations among results from replicate samples measured on a given day were within this range. Larger deviations were occasionally observed when replicate results for multicomponent samples were obtained on different days; in some cases results from different days were averaged and their standard deviations plotted (e.g., in Figure 5D) to illustrate this dayN
to-day variation. However, no conclusions are drawn from such data sets unless supported by comparisons of data sets with small deviations ( lo-’ M) binding sites. For this model, the PDE activity is predicted by eq 1 if CT,the concentration of Tu-GTPyS, is substituted for L. Alternatively, the data were analyzed in terms of a “high-affinity model”, in which only a small fraction of the total To-GTPyS is assumed to be capable of activating PDE, but is assumed to do so with relatively high affinity (Kact C M). For this model, the PDE activity, A, at each Tu-GTPyS concentration is predicted by eq 2. In this equation,f is the fraction of active
Tu-GTPyS, CTi s the concentration of added Tu-GTPyS,and Stis the total concentration of sites for Tu-GTPyS binding to PDE. Stis assumed to be equal to twice the concentration of activatable PDE, which was determined from the PDE activity, Amax,observed at saturating CT.The parameter b is defined as b E - St - ~CT). Values f o r 5 Amax,and Kactwere determined by a nonlinear least-squares fitting procedure. A first approximationfor A, was obtained from the mean activity at saturating CT,and the first approximation offwas obtained by usingf= AmaxSt/CT at the equivalencepoint. The equivalencepoint was estimated graphically as the intersection of two straight lines, one drawn through the ascending, linear portion of thedata and the other drawn horizontal to the x-axis through the mean activity value at saturating CT. Kact was then estimated by systematically varying its value until a minimum value of x2 was obtained.
Biochemistry, Vol. 31, No. 39, 1992 9505
T,-GTPyS and Phospholipid Activation of cGMP PDE
r l t c ="
p!, 1, '1, ,I
,
i
T
,
.
,
PCLUV
.
i
I
cGMP
cGMP
i 0 . 04
,]
0
2
TIME (minutes)
4 0
cGMP
J.
, ,
, ,
2
TIME (minutes)
4 0
c
:, , , ,] 2 4
o
lO O
15
Further minimization of x2 was obtained by an iterati\ e procedure in which all three parameters (f, Kact and Amax) were varied until further iterations did not result in a lower value of x2. The final best fit values for f and Amax never varied from the original estimates by more than 2% of the final values. Thus, in this model f and Amaxwere very well determined by the data. The values reported in Table I are the best fit values &uf or uA,calculated numerically as uf = ( 2 / d 2 ~ 2 / d f 2 ) )or 1 / 2U A = ( 2 / ( d 2 ~ 2 / d A m a x 2 ) (Bevington, )1/2 1969). Because the value of Kact was much lower than the values of f c T needed to elicit measurable PDE activation, the x2 surface is very asymmetric: x2 is very sensitive to increases in Kact above the best fit value but very insensitive to decreases in Kact. Therefore a meaningful value of Q could not be calculated, and only an upper limit could be reliably determined for Kact. The values reported in Table I are those giving the minimum x2values, and the upper limit is taken as that value which resulted in a x2 value 50% higher than the minimum value. Equation 2 was also used to analyze GTPyS activation of transducin and PDE in native ROS membranes (Figure 5B), with CT replaced by CG, the concentration of GTPyS, f replaced by 1, and St defined here as the total concentration of GTPyS-binding sites on transducin, determined by fitting, and b 1 (-Kact - St - CG). The assumptions implicit in this treatment are that PDE in ROS is maximally activated (i.e., A = Amax) when all T, has bound GTPyS, but that, again, only a fraction of the T,-GTPyS formed [in this case given by f = (2Am,/4200 s-l)/St] is competent to activate PDE, and that the dissociation constant for GTPyS binding to T, is lower than Kact. Equations 1 and 2 are equivalent under conditions where the concentration of titrant, f c T or f c G , is much greater than St, the concentration of binding sites. Direct measurements of PDE binding to membranes (Figure 6) were fit to theoretical curves with one binding constant (eq 1 with bound PDE substituted for A) or two binding constants: B = {BmaxlL/(L+ nKpL1)) + {(1OO-B~axl)L/(L + ~KPLJI (3) where B represents the percentage of membrane-bound PDE at each phospholipid concentration (L). Bmxland nKpLvalues were determined from the fit, with the constraint that Bmaxl
45
60
75
[PHOSPHOLIPID] (pM)
TIME (minutes)
FIGURE 1: Reconstitutionof T.-GTPyS-stimulated PDE activity on phospholipidvesicles. cGMPhydrolysisresulting from the activation of 10 nM PDE by 0.6 pM T,-GTPyS in the presence of 35 pM PC LUV was assayed by the pH method. The final components of each assay mixture were the same but the order of addition varied. (A) PC LUV were added to T,-GTPyS and PDE. (B) PDE was added to T,-GTPrS and PC LUV. (C) T,-GTPyS was added to PC LUV and PDE. TheT,-GTPyS used in this experiment was the preparation of Figure 5A.
30
FIGURE2: Phospholipid dependence of T,-GTPyS-stimulated PDE activation. Titration of 10 nM PDE and 0.3 pM T,-GTPyS with phospholipid membranes from PC LUV (closed squares) or U W ROS (closed circles). PDE activity was plotted as the percentage of the total trypsin-inducedactivitypre:.int in the sample (independent samples, mean SD, U W ROS, n = 2; PC LUV, n = 3). The curve was calculated from eq 1 using the values for n K p ~and A,,, given in Table 11. Activity present in the U W ROS (
